Fear Responses In Zebrafish Research Articles

β-Carboline (FG-7142) modulates fear but not anxiety-like behaviour in zebrafish

The β-Carboline FG-7142 is a partial inverse agonist at the benzodiazepine allosteric site on the GABA-A receptor that induces anxiogenic, proconvulsant, and appetite-reducing effects in many species, including humans. Seizure-kindling effects have been well studied, however anxiogenic properties are relatively unexplored. This study aimed to investigate concentration-dependent effects of FG-7142 on anxiety-like behaviour and fear responses in zebrafish (Danio rerio) using the open-field test (OF) and novel object approach test (NOA). A U-shaped distribution was found with maximal responses in increased immobility and reduced distance moved at 10 µM in the NOA but not the OF. Follow up experiments demonstrated a lack of effect in repeated OF testing and no changes in opercular movements. Furthermore, the effect of FG-7142 was reversed with ethanol treatment. These results suggest that FG-7142 elicits a ‘freezing’ response in zebrafish via the introduction of novelty, suggesting fear-induction. These findings indicate that FG-7142 may act as an agent to promote acute fear responses in zebrafish.

The Tapping Assay: A Simple Method to Induce Fear Responses in Zebrafish.

The zebrafish is increasingly employed in behavioral neuroscience as a translationally relevant model organism for human central nervous system disorders. One of the most prevalent CNS disorders representing an unmet medical need is the disorder cluster defined under the umbrella term anxiety disorders. Zebrafish have been shown to respond to a variety of anxiety and fear inducing stimuli and have been suggested for modeling human anxiety. Here, we describe a simple method with which we intend to induce fear/anxiety responses in this species. The method allows us to deliver a visual and lateral line stimulus (vibration or "tapping") to the fish with the use of a moving object, a ball colliding with the side glass of the experimental tank. We describe the hardware construction of the apparatus and the procedure of the behavioral paradigm. We also present data on how zebrafish respond to the tapping. Our results demonstrate that the method induces significant fear/anxiety responses. We argue that the simplicity of the method and the efficiency of the paradigm should make it popular among those who plan to use zebrafish as a tool in anxiety research.

Nicotine increases fear responses and brain acetylcholinesterase activity in a context-dependent manner in zebrafish

Nicotine is an alkaloid with positive effects on learning and memory processes. Exposure to conspecific alarm substance (CAS) elicits fear responses in zebrafish, but the effects of nicotine on aversive behaviors and associative learning in this species remain unclear. Here, we evaluated whether nicotine enhances contextual fear responses in zebrafish and investigated a putative involvement of brain acetylcholinesterase (AChE) in associative learning. Fish were exposed to 1 mg/L nicotine for 3 min and then kept in non-chlorinated water for 20 min. Later, animals were transferred to experimental tanks in the absence or presence of 3.5 mL/L CAS for 5 min (training session). After 24 h, fish were tested in tanks with similar or altered context in the absence of CAS (post-training session) and brain AChE activity was further assessed. At training, CAS increased freezing, erratic movements, and decreased the time spent in top area, while nicotine abolished the effects of CAS on erratic movements. Nicotine/CAS group tested in a similar context showed exacerbated freezing and reduced transitions to top area. Moreover, a decrease in distance traveled was observed in control, nicotine, and nicotine/CAS groups at post-training. Nicotine also stimulated brain AChE activity in CAS-exposed animals reintroduced in tanks with similar context. Although freezing bouts and time spent in top could serve as behavioral endpoints that reflect CAS-induced sensitization, the effects of nicotine occurred in a context-dependent manner. Collectively, our data suggest an involvement of cholinergic signaling in aversive learning, reinforcing the growing utility of zebrafish models to explore the neurobehavioral effects of nicotine in vertebrates.

Granule cells control recovery from classical conditioned fear responses in the zebrafish cerebellum

Although previous studies show that the cerebellum is involved in classical fear conditioning, it is not clear which components in the cerebellum control it or how. We addressed this issue using a delayed fear-conditioning paradigm with late-stage zebrafish larvae, with the light extinguishment as the conditioned stimulus (CS) and an electric shock as the unconditioned stimulus (US). The US induced bradycardia in the restrained larvae. After paired-associate conditioning with the CS and US, a substantial population of the larvae displayed CS-evoked bradycardia responses. To investigate the roles of the zebrafish cerebellum in classical fear conditioning, we expressed botulinum toxin or the Ca2+ indicator GCaMP7a in cerebellar neurons. The botulinum-toxin-dependent inhibition of granule-cell transmissions in the corpus cerebelli (CCe, the medial lobe) did not suppress the CS-evoked bradycardia response, but rather prolonged the response. We identified cerebellar neurons with elevated CS-evoked activity after the conditioning. The CS-evoked activity of these neurons was progressively upregulated during the conditioning and was downregulated with repetition of the unpaired CS. Some of these neurons were activated immediately upon the CS presentation, whereas others were activated after a delay. Our findings indicate that granule cells control the recovery from conditioned fear responses in zebrafish.

Reproductive Neuroendocrine Pathways of Social Behavior.

Social behaviors are key components of reproduction, because they are essential for successful fertilization. Social behaviors, such as courtship, mating, and aggression, are strongly associated with sex steroids, such as testosterone, estradiol, and progesterone. Secretion of sex steroids from the gonads is regulated by the hypothalamus–pituitary–gonadal (HPG) axis in vertebrates. Gonadotropin-releasing hormone (GnRH) is a pivotal hypothalamic neuropeptide that stimulates gonadotropin release from the pituitary. In recent years, the role of neuropeptides containing the C-terminal Arg–Phe–NH2 (RFamide peptides) has been emphasized in vertebrate reproduction. In particular, two key RFamide peptides, kisspeptin and gonadotropin-inhibitory hormone (GnIH), emerged as critical accelerator and suppressor of gonadotropin secretion. Kisspeptin stimulates GnRH release by directly acting on GnRH neurons, whereas GnIH inhibits gonadotropin release by inhibiting kisspeptin, GnRH neurons, or pituitary gonadotropes. These neuropeptides can regulate social behavior by regulating the HPG axis. However, distribution of neuronal fibers of GnRH, kisspeptin, and GnIH neurons is not limited within the hypothalamus, and the existence of extrahypothalamic neuronal fibers suggests direct control of social behavior within the brain. It has traditionally been shown that central administration of GnRH can stimulate female sexual behavior in rats. Recently, it was shown that Kiss1, one of the paralogs of kisspeptin peptide family, regulates fear responses in zebrafish and GnIH inhibits sociosexual behavior in birds. Here, we highlight recent findings regarding the role of GnRH, kisspeptin, and GnIH in the regulation of social behaviors in fish, birds, and mammals and discuss their importance in future biological and biomedical research.

Animated bird silhouette above the tank: Acute alcohol diminishes fear responses in zebrafish

Alcohol dependence and alcohol abuse represent major unmet medical needs. The zebrafish is considered to be a promising vertebrate species with which the effects of alcohol on brain function and behavior and the mechanisms underlying these effects may be studied. Alcohol is known to induce alterations in motor function as well as fear and anxiety. Here we utilize a recently developed fear paradigm in which we employ an animated (moving) image of a bird silhouette. We measure the effect of acute alcohol administration (dose range employed: 0.00–0.75vol/vol percentage, bath exposure for 60min) on the behavioral responses of zebrafish. We test these responses during a pre-stimulus, stimulus and post-stimulus period of the task using both a video-tracking and an observation based quantification method. The fear inducing stimulus was found to decrease the distance of the zebrafish from the bottom of the tank, to increase number of erratic movements, and to increase the number of jumps in alcohol exposed fish (versus control fish). Alcohol attenuated these fear responses in a dose dependent manner. In addition, alcohol decreased general activity at the highest dose, an effect that was independent of the presentation of the stimulus. We discuss the similarities and differences between observation and video-tracking based results and conclude that fear paradigms will be useful in revealing alcohol induced functional changes in the brain of zebrafish.

Effects of animated images of sympatric predators and abstract shapes on fear responses in zebrafish

Previously, zebrafish were shown to respond to the presence of a live predator, the sight of a live predator, moving computer images of a predator, and images of different moving objects in a context and stimulus specific manner. For example, zebrafish showed a robust antipredatory reaction towards a sympatric predator, the Indian leaf fish (Nandus nandus) but not towards an allopatric predator or sympatric or allopatric harmless fish species. In a subsequent study, however, we found zebrafish not to show avoidance in response to the image of another sympatric predator, Xenentodon cancila. The results implied that zebrafish may possess genetic predispositions that allow them to effectively evade at least certain sympatric predators without any prior exposure to them. However, it is not known whether zebrafish can respond to other sympatric predators with antipredatory reactions without any prior exposure to such predators. It is also not known whether such responses may be dependent upon, or idiosyncratic to, predator species. To answer these questions, we examined the effect of computer animated (moving) images of five different predator species all sympatric with zebrafish (Nandus nandus, Channa maculata, Xenentodon cancila, Mystus bleekeri, Chitala chitala) and of two abstract shapes (star and rectangle, also animated) on zebrafish behaviour. The results confirmed that the sight of N. nandus is an aversive stimulus and that the image of X. cancila is not. However, the results also revealed that images of three other species (C. maculata, M. bleekeri, C. chitala) and that of a rectangle induce even more robust aversive reactions than what is elicited by N. nandus. At this point we do not know what features of the images may be perceived as aversive by zebrafish or whether the observed behavioural responses would be adaptive or effective in nature. Nevertheless, we argue that image-presentation based fear paradigms will be useful in future translational research. We also conclude that it may be premature to decide whether zebrafish can recognize their sympatric predators.

The habenula is crucial for experience-dependent modification of fear responses in zebrafish

The zebrafish dorsal habenula (dHb) shows conspicuous asymmetry in its connection with the interpeduncular nucleus (IPN) and is equivalent to the mammalian medial habenula. Genetic inactivation of the lateral subnucleus of dHb (dHbL) biased fish towards freezing rather than the normal flight response to a conditioned fear stimulus, suggesting that the dHbL-IPN pathway is important for controlling experience-dependent modification of fear responses.